Human cytomegalovirus (HCMV) is a ubiquitous and significant pathogen that stimulates cell cycle progression. HCMV is a cofactor for cardiovascular diseases with significant proliferative components such as coronary restenosis and transplant vascular sclerosis (TVS), and may play a role in certain types of cancers, most notably, malignant gliomas (GBMs). Cellular proliferation and transformation is regulated by the retinoblastoma family of tumor suppressors (Rb, p107, and p130). The Rb proteins, in conjunction with the E2F family of transcription factors, regulate the expression of cellular genes required for DNA replication such as nucleotide biosynthetic enzymes (NBEs) of the de novo pathway, as well as secreted factors involved in wound healing and angiogenesis. The expression of E2F-responsive genes is inhibited when the hypophosphorylated forms of the Rb proteins are bound to the E2F proteins. Degradation of the hypophosphorylated Rb proteins, or their hyperphosphorylation disrupts their complexes with E2F, thus allowing free E2F to activate gene expression. HCMV encodes four proteins that modulate the Rb-E2F pathway: pp71, IE1, IE2, and the focus of this application, UL97. Because HCMV is associated with proliferative diseases and encodes multiple proteins that modulate the Rb-E2F pathway, we hypothesize that the Rb-E2F pathway plays a critical role in HCMV replication and pathogenesis. Here we propose to explore the roles of the Rb, E2F, and UL97 proteins during HCMV infection. UL97 is a member of a family of conserved herpesvirus protein kinases (CHPKs) that directly phosphorylates the Rb protein and inactivates it, leading to the induction of cellular E2F-responsive genes. We hypothesize that some of these E2F-responsive gene products (the NBEs) are responsible for the UL97-mediated stimulation of viral DNA replication. We further speculate that other E2F-responsive gene products induced by UL97 (secreted wound healing and angiogenesis factors) may in turn be partially responsible for proliferative pathologies associated with HCMV infection, such as restenosis, TVS, and GBMs. The long-term goal of this project is to determine how Rb inactivation by UL97 and other HCMV proteins impacts cell cycle progression, HCMV replication, and viral pathogenesis in both cell autonomous and non-cell-autonomous manners, and to use this information to design therapies to treat proliferative diseases associated with HCMV infection. PHS 398/2590 (Rev. 05/01) Page 1 Continuation Format Page